The StrataPrep total RNA microprep kit provides a
convenient method for isolating total RNA from small samples of cells. For
the purification, RNA is bound to a solid support, which eliminates the
need for organic extraction and alcohol precipitation. The resulting RNA
is high quality, with no detectable DNA contamination and is suitable for
subsequent quantitative or conventional RT-PCR.

Stratagenes recently released StrataPrep total RNA miniprep kit is
designed to isolate total RNA from cells ranging from 105 to 107
cells. However, when you need total RNA from a smaller sample, the
StrataPrep total RNA microprep kit is ideal. In the past, isolating total
RNA from very small samples by traditional methods was difficult and inefficient
because yields from small samples tend to be low due to losses during
phenol-chloroform extraction and/or ethanol precipitation. Additionally, organic
solvents are hazardous. Now, with Stratagenes StrataPrep total RNA
microprep kit, total RNA can be extracted from as few as 10 cells to as many as
5 x 105 cells.

Easy Protocol

Fig.1

As shown in the Figure
1 overview, cultured cells are disrupted by guanidine thiocyanate,
then bound to a silica-based fiber matrix within a spin cup. RNA
is immobilized on the fiber matrix, which allows contaminants to be removed
while avoiding organic extractions and ethanol precipitation. DNA is removed
with a single DNase I incubation step directly on the matrix, the matrix
is washed, and pure RNA is eluted with a small volume of buffer. The recovered
RNA is then ready to use. The StrataPrep total RNA microprep kit provides
all nece
ssary buffers and components for total RNA purification including
lyophilized DNase I for convenient room temperature storage.

Isolating RNA from Different Sources

Average yields of RNA from various cells

Cell line

5 x 105 cells

1 x 105 cells

Yield (g)

A260/A280

Yield (g)

A260/A280

HeLa

10.1

2.0

2.1

2.0

HL-60

2.8

2.1

0.5

2.1

THP-1

4.1

2.0

0.7

2.1

NIH/3T3

7.3

2.0

1.5

2.1

CHO

5.7

2.0

1.2

2.1

Table
1 and Table
2 show total RNA yields from varying cell numbers of different cell
types. A typical mammalian cell contains approximately 10-5
g of total RNA.1 The yields for all cell lines are close to
or higher than this number, indicating high yields. The A260/A280
ratios measured for all samples isolated from 5 x 105 or 1
x 105 cells were 2.0 to 2.1, indicating high-quality RNA.

Average Yields of RNA* from Smaller Numbers of Cells

Cell line

104 cells

103 cells

102 cells

101 cells

Yields (ng)

HeLa

243

28

ND**

0.4

HL-60

86

8.2

0.8

0.2

THP-1

88

10.3

0.8

0.3

NIH/3T3

176

17.0

1.1

1.0

CHO

154

12.5

1.4

0.5

* RNA was quantitated by RiboGreen assay.
Isolations were performed in duplicate.
** Not done

Total RNA from smaller samples (Tab
le
2) was quantitated in fluorescence assays using the RiboGreen
RNA quantitation kit (Molecular Probes) since absorbance measurements
are not sensitive enough for accurate quantitation. The quantity
of RNA isolated from 10 cells is at or near the detection limit for RiboGreen
(1 ng/ml as per manufacturer). Overall, RNA isolated from 104
to 101 cells reflects yields of approximately 10-5
g of total RNA per cell, although yield generally varies according to
cell size.

Representative RNA samples isolated from 5 x 105 cells (Table
1) were electrophoresed in formaldehyde-agarose gels to check the
integrity of the RNA. All of the samples are intact, according to this
analysis, as observed by the distinct ribosomal RNA bands (Figure
2).

Fig.2

RT-PCR Analysis of RNA

The high quality of total RNA isolated from 5 x 105 cells
was confirmed by successful RT-PCR amplification of a relatively long
target. Primers specific for replication factor C (RFC) were used in RT-PCR
(Figure
3) and the expected 3.1-kb band was observed. It has been reported
that RFC RNA undergoes alternative processing, which leads to mRNA of
other sizes.4 This may be the case for RNA from HeLa,
HL-60, and CHO cells since additional PCR products are evident in these
samples.

Fig.3

Total RNA from 5 x 105 cells was also tested for the presence
of contaminating DNA by RT-PCR, with and without reverse transcriptase.
This assay uses GAPDH primers that amplify a 0.2-kb fragment from
mRNA and genomic DNA. There are at l
east 400 copies of GAPDH pseudogenes
in the genomes of the mouse and rat and approximately 20 copies in the
human genome.5,6Figure
4 shows that DNA contamination is not detectable as demonstrated by
the lack of product for reactions in which reverse transcriptase was omitted.

Fig.4

RT-PCR Analysis of RNA from Small Cell Numbers

Total RNA isolated from 105, 104, 103,
102, and 101 HeLa cells was tested as template in
RT-PCR. Amplification using the human GAPDH primer set for RT-PCR
with the prostar Ultra HF RT-PCR system7 was
successful with all samples, including RNA isolated from only 10 cells
(Figure
5). For reactions where reverse transcriptase or cDNA template was
omitted, no bands were detected; therefore, the target sequence DNA did
not contaminate the reactions.

Fig.5

Detecting RNA with Quantitative RT-PCR

RNA from varying numbers of HeLa and THP-1 cells was also subjected to
real-time quantitative-RT-PCR analysis using a GAPDH-specific molecular
beacon for detection. The molecular beacon hybridizes to the target sequence
and emits fluorescence in this conformation. The threshold cycle number
(Ct) is inversely proportional to the concentration of target
sequence in the reaction.8 The Mx4000 molecular
beacon GAPDH expression analysis kit specifically detects GAPDH mRNA sequences
but not genomic DNA or pseudogene sequences.9 Detecting
as little as 1 pg of human total RNA or 0.01 pg of poly(A)+
RNA is possible with this kit.10 Signal was readily det
ected
from one tenth of the RNA sample isolated from a one-cell equivalent,
with Ct values of 35 (HeLa) and 36 (THP-1) (Figure
6). As expected, the Ct value increases with a decrease
in cell number.

Fig.6

Conclusions

The StrataPrep total RNA microprep kit is the method of choice for isolating
RNA from small cell samples. The protocol and supplied reagents provide a method
to purify pure, intact RNA with high yields. Amplification by conventional
RT-PCR is feasible from as few as 1 cell and by quantitative RT-PCR from only
one cell.

Acknowledgments

The authors thank Robert Saiz and Reinhold Mueller for performing molecular
beacon RT-PCR and Sylvia Norman for assisting with conventional RT-PCR.

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